Chronic hyperammonemia prevents changes in brain energy and ammonia metabolites induced by acute ammonium intoxication.

Acute ammonia toxicity has been attributed to the depletion of energy metabolite intermediates. Ingestion of an ammonium containing diet produces hyperammonemia and protects rats against acute ammonium intoxication. We have tested the effect of chronic hyperammonemia on the brain contents of energy and ammonia metabolite intermediates and on the effect on these contents of acute ammonia intoxication (i.p. injection of 7 mmol/kg of ammonium acetate). Chronic hyperammonemia was induced in rats by feeding them a diet containing 20% ammonium acetate. Control rat were fed the same diet without addition of ammonium acetate. It is shown that chronic hyperammonemia did not affect the content of most metabolites, the only remarkable changes are the increases of the contents of ammonia (46%), glutamine (81%), acetoacetate (31%) and of the mitochondrial NAD+/NADH ratio (32%) as well as the marked decrease of beta-hydroxybutyrate (by 86%). Chronic hyperammonemia prevents most changes in metabolites induced by acute ammonium intoxication (i.p. injection of 7 mmol/kg of ammonium acetate). In control rats it was a marked breakdown of glycogen and increased contents of glucose, lactate and pyruvate, with decreased cytosolic NAD+/NADH ratio and beta-hydroxybutyrate and ATP contents. These changes were nearly completely prevented in hyperammonemic rats. In controls, ammonia increased 12.8-fold while glutamate and aspartate decreased by approximately 40% and glutamine and alanine raised by 37% and 93%, respectively; in hyperammonemic rats ammonia increased 6.9-fold while glutamate, glutamine and alanine were not significantly affected. Also the mitochondrial NAD+/NADH ratio raised by 18-fold in controls and by 6-fold in hyperammonemic rats. These results indicate that chronic hyperammonemia markedly prevents the alterations of the contents of energy and ammonia metabolites induced by acute ammonium intoxication.

Sustained recovery of Na(+)-K(+)-ATPase activity in sciatic nerve of diabetic mice by administration of H7 or calphostin C, inhibitors of PKC.

We have previously shown that intraperitoneal injection of H-7, an inhibitor of PKC, restores completely the activity of Na(+)-K(+)-ATPase in sciatic nerve of diabetic mice; however, the effect was transient, with a half-life of approximately 1 h under the conditions used. This work assessed whether calphostin C, a new more potent and specific inhibitor of PKC, is also able to restore the activity of Na(+)-K(+)-ATPase in sciatic nerve of ALX-induced diabetic mice and also assessed if continuous administration of H-7 or calphostin C can afford sustained recovery of the ATPase. Small amounts of calphostin C (i.e., 2 micrograms/kg) restore entirely the activity of the enzyme. Larger doses (e.g., 30 micrograms/kg) can be administered with equal results. The ED50 was approximately 0.5 micrograms/kg. This indicates that calphostin C is approximately 20,000 times more potent than H-7 in restoring the ATPase activity in diabetic mice. A single intraperitoneal injection of 1 or 10 micrograms/kg of calphostin C maintains the enzyme for 4 and 8 h, respectively. Administration of H-7 by continuous delivery from micro-osmotic pumps implanted in the back of the mice maintains the Na(+)-K(+)-ATPase for 24 h, although the activity decreases thereafter. This is the result of instability of H-7 in solution. Continous administration of calphostin C maintains the activity of the ATPase at nearly normal values for at least 2 wk. The results support the hypothesis that, in sciatic nerve tissue of diabetic animals, the activity of PKC is increased, leading to higher phosphorylation of Na(+)-K(+)-ATPase, which results in the decreased activity observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Prenatal exposure of rats to ammonia impairs NMDA receptor function and affords delayed protection against ammonia toxicity and glutamate neurotoxicity.

The aim of this work was to assess whether perinatal hyperammonemia impairs the function of NMDA receptors and whether this impairment affords protection against acute ammonia toxicity and glutamate and NMDA neurotoxicity. Rats were exposed to ammonia during the prenatal and lactation periods by feeding the female rats an ammonium-containing diet since day 1 of pregnancy. After weaning (at postnatal day 21), the pups were fed a normal diet with no ammonia added. This treatment resulted in a marked decrease of the growth rate of the animals, which was maintained even 1 month after normalization of ammonia levels. Rats exposed to ammonia were more resistant than controls to acute ammonia toxicity 13 days after feeding a normal diet but not at 3 months. Primary cultures of cerebellar neurons from hyperammonemic rats showed decreased binding of [3H]MK-801 and were remarkably more resistant than controls to glutamate and NMDA toxicities. Also, the increase in aspartate aminotransferase activity induced by low concentrations of NMDA was not produced in such cultures. These results indicate that exposure to ammonia during the prenatal and lactation periods results in long-lasting impairment of NMDA receptor function. This would be the reason for the delayed protection afforded by exposure to low ammonia levels against acute ammonia toxicity in animals and against glutamate and NMDA toxicity in neuronal cultures.

Actinomycin D decreases protein kinase C content and induces neuritogenesis in neuroblastoma cells.

We have previously reported that inhibition of protein kinase C induces differentiation of neuroblastoma cells in culture. It is shown now that actinomycin D, a well known inhibitor of DNA synthesis, reduces selectively the content of protein kinase C and induces neuritogenesis in Neuro 2a cells in culture.

Inhibition of protein kinase C induces differentiation of neuroblastoma cells.

It is shown that 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), a specific inhibitor of protein kinase C, induces neuritogenesis in neuro 2a cells. The percentage of differentiated cells was 9%, 20%, 59% and 85% at 0, 17, 85 and 500 microM H7, respectively. The number of neurites cell increased 2-, 8- and 14-fold over the controls for 17, 85 and 500 microM H7, respectively. These results indicate that protein kinase C plays a key role in the control of differentiation of neural cells and that its specific inhibition may be of basic as well as of practical importance.

Inhibition of protein kinase C arrests proliferation of human tumors.

We have shown that inhibition of protein kinase C by 1-5-isoquinolinylsulfonyl-2-methylpiperazine, H7, induces differentiation and inhibits proliferation of Neuro 2a cells. We have now tested if H7 is able to inhibit proliferation of: 1) human tumor cell lines from tissues other than brain; and 2) primary cultured cells from several human brain tumors. H7 inhibits, in a dose-dependent manner, proliferation of all human tumor cell lines tested and of primary cultured cells from human brain tumors. These results indicate that inhibition of protein kinase C inhibits proliferation of tumoral cells, therefore, H7, and likely other inhibitors of protein kinase C, could be useful in the clinical treatment of brain (and probably other) tumors.

Long-term ingestion of ammonium inhibits lysosomal proteolysis in rat liver.

A standard diet was supplemented with ammonium acetate (20%, w/w). The effect on liver protein degradation of oral administration of the ammonium diet to rats for 6 weeks has been studied. It is shown that lysosomal proteolysis is markedly decreased (by 62%) while non-lysosomal proteolysis is inhibited by 11%. This is the first report showing that ammonium ingestion inhibits liver proteolysis.

Acute ammonia toxicity is mediated by the NMDA type of glutamate receptors.

Previous experiments in our laboratory suggested that ammonium toxicity could be mediated by the NMDA type of glutamate receptors. To assess this hypothesis we tested if MK-801, a specific antagonist of the NMDA receptor, is able to prevent ammonium toxicity. Mice and rats were injected i.p. with 12 and 7 mmol/kg of ammonium acetate, respectively. 73% of the mice and 70% of the rats died. However, when the animals were injected i.p. with 2 mg/kg of MK-801, 15 min before ammonium injection, only 5% of the mice and 15% of the rats died. The remarkable protection afforded by MK-801 indicates that ammonia toxicity is mediated by the NMDA receptor.

Hyperammonemia decreases body fat content in rat.

We have developed an animal model of hyperammonemia consisting of feeding rats a diet containing 20% (w/w) ammonium acetate. Ingestion of this diet markedly affects carcass composition, with a 46% reduction in lipid content. The ammonium diet alters levels of several key compounds involved in lipid metabolism. Long-chain acylcarnitine is increased in liver by approx. 60% while free carnitine and acetylcarnitine are unaffected. The hepatic content of acetyl-CoA increases by approx. 50%. The level of ketone bodies in blood increases by 32% but remains unchanged in liver. Our data indicate that hyperammonemia alters lipid metabolism and results in a significant decrease in body lipid content.

H7, a protein kinase C inhibitor, increases the glutathione content of neuroblastoma cells.

It is shown that the intracellular glutathione (GSH) concentration of neuroblastoma-2a cells in culture increases with a maximum at 24 h after starting treatment with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), an inhibitor of protein kinase C (PKC). Other inhibitors of this and other protein kinases, e.g. sphingosine, staurosporine, and HA 1004, at the concentrations tested, had a less marked or negligible effect on intracellular GSH concentration. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was also tested and showed no significant effect 24 h after addition.

H7, an inhibitor of protein kinase C, inhibits tumour cell division in mice bearing ascitic Ehrlich's carcinoma.

We have previously shown that H7, an inhibitor of protein kinase C (PKC), inhibits proliferation of several cell lines as well as of primary cultured cells from human tumours. The aim of this work was to assess whether H7 is able to prevent the division of tumour cells in mice bearing Ehrlich's ascitic carcinoma. The LD50 of H7 injected intravenously was 61 mg/kg and 94 mg/kg for starved and fed mice, respectively. Acute intraperitoneal injection of 100 mg/kg of H7 decreased the number of mitoses in tumoral cells from ascitic fluid of mice bearing the carcinoma. The reduction was maximal (approximately 50%) after 90 min and then the number of mitosis rose due to a decrease in H7. Continuous delivery of H7 from mini-osmotic pumps implanted on the backs of the mice reduced the number of mitoses by approximately 65%, and the effect was maintained for approximately 24 h. The effect cannot be maintained for longer because H7 is unstable at body temperature. These results indicate that inhibition of PKC can block division of tumour cells in carcinoma-bearing animals, and support the idea that inhibitors of PKC could be useful for the clinical control of proliferation of certain tumours.

Nicotine prevents glutamate-induced proteolysis of the microtubule-associated protein MAP-2 and glutamate neurotoxicity in primary cultures of cerebellar neurons.

The aim of this work was to assess whether nicotine prevents glutamate neurotoxicity in primary cultures of cerebellar neurons, to try to identify the receptor mediating the protective effect and to shed light on the step of the neurotoxic process which is prevented by nicotine. It is shown that nicotine prevents glutamate and NMDA neurotoxicity in primary cultures of cerebellar neurons. The protective effect of nicotine is not prevented by atropine, mecamylamine or dihydro-beta-erythroidine, but is slightly prevented by hexamethonium and completely prevented by tubocurarine and alpha-bungarotoxin, indicating that the protective effect is mediated by activation of alpha7 neuronal nicotinic receptors. Moreover, alpha-bungarotoxin potentiates glutamate neurotoxicity, suggesting a tonic prevention of glutamate neurotoxicity by basal activation of nicotinic receptors. Nicotine did not prevent glutamate-induced rise of free intracellular calcium nor depletion of ATP. Nicotine prevents glutamate-induced proteolysis of the microtubule-associated protein MAP-2 and disaggregation of the neuronal microtubular network. The possible mechanism responsible for this prevention is discussed.

Protein kinase C isoforms and cell proliferation in neuroblastoma cells.

The expression of protein kinase C isoforms in the neuroblastoma cell line Neuro 2a has been studied. It is shown that Neuro 2a cells express alpha, delta, epsilon and zeta PKCs. Inhibition of cell proliferation by using protein kinase C inhibitors (H7 or calphostin C) or medium without glutamine affects markedly the pattern of PKC isoforms. All treatments reduced significantly (50-70%) the content of PKC alpha. None of the treatments altered PKC zeta or epsilon. The content of PKC delta was increased (88-120%) in cells treated with PKC inhibitors but was slightly reduced in cells incubated in medium without glutamine. However, none of the treatments affected the content of the corresponding mRNAs. Long-term treatment of synchronized cells with the phorbol ester PMA depletes PKC alpha but not PKC delta or zeta and only partially PKC epsilon. This treatment with PMA did not affect DNA synthesis, indicating that PKC alpha does not play a significant role in the control of proliferation of these cells.

Differential effects of the protein kinase C inhibitors H7 and calphostin C on the cell cycle of neuroblastoma cells.

We have studied the effect of protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) and calphostin C on the cycle of Neuro-2a cells. Both compounds inhibited cell proliferation and DNA synthesis. Transition from G2 to M phase was not altered by these compounds. Calphostin C blocked the cells in G0/G1, while H7 did not at any specific point in the cell cycle. We also show that the antiproliferative effect induced by both inhibitors is reversible.

Inhibitors of protein kinase C prevent the toxicity of glutamate in primary neuronal cultures.

Glutamate-induced neurotoxicity has been proposed to depend on a sustained increase of intracellular free Ca2+ levels. However, the molecular mechanism(s) involved are not well understood. Some results suggest that activation of protein kinase C by the increased levels of Ca2+ could play a role in the mediation of glutamate neurotoxicity. To assess this hypothesis we have tested if the 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H7) and calphostin C, inhibitors of protein kinase C, are able to protect neurons in primary culture from glutamate-induced cell death. It is shown that both H7 and calphostin C prevent nearly completely the death of neurons from cerebellum, even when 2 mM glutamate was used. HA-1004, an inhibitor of cyclic nucleotide-dependent protein kinases, did not protect neurons. The protective effect was maximum at approximately 10 microM H7 and at approximately 10 nM calphostin C. The results reported support the hypothesis that protein kinase C plays a key role in the mediation of glutamate neurotoxicity.

Protein kinase C inhibitors, H7 and calphostin C, inhibit induction of DNA synthesis by cytosolic extracts of exponentially growing neuroblastoma cells in isolated nuclei.

Cytoplasmic extracts from proliferating Neuro-2a cells contain a protein factor, ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. Cytoplasmic extracts derived from quiescent-made Neuro-2a cells contain none or very little ADR activity, but this activity can be generated after a brief exposure of cytosolic extracts to a membrane-enriched fraction derived from exponentially growing Neuro-2a cells. ADR activity appears at the beginning of the S phase of the cell cycle. Moreover it appears to be a protease, because aprotinin inhibits ADR activity. ADR activity can be also inhibited by the protein kinase C inhibitors, 1-(5-isoquinoline-sulfonyl)-2- methylpiperazine (H7) and calphostin C.

Lack of correlation between glutamate-induced depletion of ATP and neuronal death in primary cultures of cerebellum.

The aim of this work was to identify, using primary cultures of cerebellar neurons, the receptors involved in glutamate-induced depletion of ATP and to assess whether there is a correlation between glutamate-induced ATP depletion and neuronal death. Glutamate induced a rapid depletion of ATP (40% decrease at 5 min). After 60 min incubation with 1 M glutamate ATP content decreased by 60-70%. Similar effects were induced by glutamate, NMDA and kainate while quisqualate, AMPA or trans-ACPD did not affect significantly ATP content. The EC50 were approximately 6, 25 and 30 microM for glutamate, NMDA and kainate, respectively. DNQX and AP-5, competitive antagonists of kainate and NMDA receptors, respectively, prevented in a dose-dependent manner the glutamate-induced depletion of ATP. These results indicate that glutamate-induced depletion of ATP is mediated by activation of kainate and NMDA receptors. Glutamate-induced neuronal death was prevented by MK-801, calphostin C, H7, carnitine, nitroarginine and W7. However, only MK-801 and W7 prevented glutamate-induced depletion of ATP, while calphostin C, H7, carnitine and nitroarginine did not. This indicates that there is not a direct correlation between ATP depletion and neuronal death.

H7, an inhibitor of protein kinase C, prevents serum-induced phosphorylation of Raf and MAP kinase in neuroblastoma cells.

We have previously shown that 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), an inhibitor of protein kinase C, inhibits proliferation of neuroblastoma cells in culture. We have now tested whether the effect of H7 is mediated by MAP kinase and Raf. It is shown that, in Neuro 2a cells, activation of protein kinase C by addition of 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (PMA), leads to phosphorylation of Raf and Mitogen-activated protein kinase (MAP kinase). PMA-induced phosphorylation of these proteins is prevented by H7. When quiescent Neuro 2a were stimulated to proliferate by addition of serum, Raf and MAP kinase were rapidly phosphorylated. Serum-induced phosphorylation of Raf and MAP kinase is prevented by H7. These results suggest that, in Neuro 2a cells, the control of proliferation by protein kinase C could be mediated by phosphorylation (and concomitant activation) of Raf and MAP kinase.

Determination of intracellular ATP in primary cultures of neurons.

The aim of this work was to develop and characterize a quick and simple procedure to determine the intracellular content of ATP in monolayer primary cultures of neurons. The baseline was to use the minimum amount of cells which still provides reproducible results. The first step consists of releasing intracellular ATP from the cells. This is accomplished by treatment with a detergent solution, the somatic cell releasing reagent from Sigma. This reagent is claimed by the manufacturer to release ATP from a suspension of viable somatic cells. The procedure has been adapted to be used for attached cells (neurons or astrocytes growing in monolayer), thus avoiding the use of alternative time-consuming procedures to release ATP such as boiling buffers or trichloroacetic acid. After its release the free ATP was measured using the firefly luciferase reaction. We have used this protocol to assess the effect of neurotoxic concentrations of glutamate on the intracellular content of ATP in neurons. The same procedure has been used successfully to determine intracellular ATP in primary cultures of astrocytes.

Prevention of the acute neurotoxic effects of phenytoin on rat peripheral nerve by H7, an inhibitor of protein kinase C.

The neurotoxic effects of a single dose of phenytoin (150 mg/kg body weight) alone or 30 min after H7 (a protein kinase C inhibitor) injection (20 mg/kg body weight) were investigated in terms of peripheral neuromuscular function and Na+,K(+)-ATPase activity of the sciatic nerve. This intraperitoneal injection of phenytoin induced complete blockade of muscle action potentials in the dorsal segmental muscles of the rat tail evoked by electric stimulation of the caudal nerve and a 40% decrease in the Na+,K(+)-ATPase activity of the rat sciatic nerve when compared with control values, measured as the difference between total and ouabain-insensitive ATPase activity. Prior administration of H7 resulted in the complete prevention of both effects. Implications of protein kinase C inhibition in phenytoin neurotoxicity are discussed.